Out-of-school sedentary behaviour (SB) was over-predicted by 109.2 (11.8) min·week−1. Predicted YAP values were within 15%–20% equivalence of the SWA estimates. The classification accuracy of the YAP MVPA estimates for compliance to 60 min·day−1 and 30 min·school-day−1 MVPA recommendations were 91%/37% and 89%/57% sensitivity/specificity, respectively. The YAP generated robust school-level estimates of MVPA and SB and has potential for surveillance to monitor compliance with PA guidelines. The accuracy of the YAP may be further improved through research with more representative UK samples to enhance the calibration process and to refine the resultant algorithms.Self-reported youth physical activity (PA) is typically overestimated. We aimed to calibrate and validate a self-report tool among English youth. Four-hundred-and-two participants  wore SenseWear Armband Mini devices (SWA) for eight days and completed the self-report Youth Activity Profile (YAP) on the eighth day. Calibration algorithms for temporally matched segments were generated from the YAP data using quantile regression. The algorithms were applied in an independent cross-validation sample, and student- and school-level agreement were assessed. The utility of the YAP algorithms to assess compliance to PA guidelines was also examined. The school-level bias for the YAP estimates of in-school, out-of-school, and weekend moderate-to-vigorous PA (MVPA) were 17.2 (34.4), 31.6 (14.0), and −4.9 (3.6) min·week Physical inactivity and sedentary behaviour (SB) are increasingly prevalent among children and young people  and are a priori hypotheses, etc.) and none of the questionnaires demonstrated acceptable reliability and validity, with content validity noted as being particularly weak [Self-report questionnaires of PA and SB overcome some of the limitations of accelerometry, by providing important information about context and setting . Moreoverly weak ,20. A varly weak . The recrly weak , but theCompared to accelerometers, there have been fewer efforts to improve the validity of self-report questionnaires to provide estimates of time spent in PA and SB. One approach is the regression-based calibration of self-report data against accelerometer-derived PA and SB estimates. This method has shown promise in estimating whole-day  and contN = 409; 212 boys). Informed parental consent and child assent were obtained from 402 participants  who were each assigned to a unique identification code. Each school received a £300 financial incentive for participating, and each participant received a £10 shopping voucher following completion of data collection, which took place between March and July 2017. The study received ethical approval from the Liverpool John Moores University Research Ethics Committee (#14/SPS/012).Eleven schools (five primary and six secondary) from northwest England were informed about the study. Nine of the 11 schools (four primary and five secondary) were recruited and students from randomly selected classes in Year  5 , Year 8  and Year 10 (15.2 ± 0.3 years) were invited to participate . Body mass was measured to the nearest 0.1 kg using digital scales . Waist circumference was measured to the nearest 0.1 cm using a non-elastic measuring tape, which was positioned around the mid-section of the waist, over the participants’ school shirts. All anthropometric measures were administered and recorded by pairs of trained researchers in accordance with standardised procedures . Height Socioeconomic status (SES) was assessed using the 2015 English Indices of Multiple Deprivation (IMD) raw scores , which wPA was measured over eight days using a SenseWear Armband Mini (SWA) . The SWA is a multi-sensor device that detects and records movement and physiological responses at 60-second epochs (default setting) to provide accurate estimates of energy expenditure. An earlier model (Sensewear Armband Pro) was used in the original US YAP calibration study . A key aSelf-reported PA and SB data were collected using the YAP . The YAPPrior to the study commencing, the YAP was minimally amended by the research team to make the clarity, language, and terminology more appropriate for English youth . Through this process, the fundamental content and meaning of the YAP questions were unaltered. Differences between the two YAP versions are highlighted in The study followed a similar protocol as that detailed in the original US YAP calibration study . Data weData were processed using an identical data processing routine to that used in the original US YAP calibration study . SWA datThe structure and questions of the YAP were designed to temporally link the SWA data to the recall responses. The first 10 questions captured a discrete time segment in which there were specific opportunities to be physically active . PreviouA key aim of our study was to validate a previously developed approach to calibration ,25. Henc−1 and out-of-school SB min·week−1, and corresponding estimates from the SWA were examined at the student level  and school level . The validity of each of the time segments was analysed at the student level using correlations and the mean absolute percentage error (MAPE), while school-level agreement was determined using the overall mean error or bias between observed and predicted values.Agreement between the US YAP algorithm-predicted in-school, out-of-school, and weekend MVPA min·weekCalibration. The analytical sample data (n = 331) were randomly allocated by school (stratified by school stage) into calibration and cross-validation data sets. The calibration data  were used to generate the YAP prediction equations for in-school, out-of-school, and weekend MVPA and out-of-school SB. SWA and English YAP data were processed identically to the previous US YAP studies [ studies ,25. Duri studies  were fit studies , tidyver studies , and mod studies .Cross-validation. Data from the remaining three schools (1 primary and 2 secondary) were used to independently assess the prediction accuracy of the English YAP algorithms from the calibration phase. Agreement was investigated by converting the YAP composite segment scores into weekly minutes of MVPA or SB using the algorithms developed in the calibration analyses. Student-level agreement for each YAP segment was determined using correlations and MAPE. Bias was calculated to explore school-level agreement. Equivalence testing was also applied with the cross-validation sample to examine whether 90% confidence intervals (CI) for YAP-predicted minutes of MVPA/SB were within a 10% range  of estimates from the SWA [ the SWA . Where tn = 331), average MVPA min·day−1 and average MVPA min·school day−1 were computed for YAP-predicted values and SWA values. Binary codes were used to classify participants according to whether they achieved at least an average of 60 min·day−1 MVPA, which reflects current PA recommendations in the UK [−1 MVPA, which reflects recommendations in the UK [To examine the potential of the English YAP algorithms for PA surveillance, the ability of the YAP to identify participants that met PA guidelines was assessed, using the SWA data as the criterion. Using the full analytical sample .The descriptive characteristics of the participants who were included in the analyses are detailed in −1 for in-school MVPA to 445.7 (106.4) min·week−1 for out-of-school SB.Correlations between US YAP-predicted and SWA estimates were weak to moderately strong, ranging from r = 0.09 (in-school MVPA) to r = 0.73 , From the three cross-validation schools, there were 129 participants with valid YAP and SWA data for at least one YAP segment of the week. Results are presented separately for each of the four segments. Student-level results are summarised in In-school MVPA. Student-level YAP predicted in-school MVPA min·week−1 and SWA-derived MVPA were weakly correlated (r = 0.11), and the MAPE was 70.8%. School-level estimates of MVPA from the YAP and SWA differed by 17.2 (34.4) min·week−1 (approximately 3.4 min·day−1). The upper and lower limits of the 90% CI for in-school YAP MVPA  were within 20% of SWA-estimated MVPA  (·week−1) .Out-of-school MVPA. The correlation between student-level out-of-school MVPA predicted by the YAP and MVPA from the SWA was r = 0.45 and the MAPE was 83.9%. The YAP over-estimated school-level MVPA compared to SWA estimates by 31.6 (28.3) min·week−1 (6.3 min·day−1), and predicted and SWA-estimated out-of-school MVPA reached agreement when the equivalence zone was set at 20% .Weekend MVPA. Weekend MVPA estimates from the YAP and SWA were moderately correlated (r = 0.52) and the MAPE was very high (199.6%). At the school-level, the YAP under-predicted weekend SWA MVPA by 4.9 (13.2) min·week−1 (−2.5 min·day−1). The 90% CI for the weekend MVPA YAP values  were within the 15% equivalence zone .Out-of-school SB. At the student-level, out-of-school time in SB was strongly correlated to SB estimated from the SWA (r = 0.80), and MAPE was 50.6%. School-level YAP-predicted out-of-school time spent in SB was 109.2 (20.5) min·week−1 (21.8 min·day−1) higher than the SWA estimates. YAP-predicted and SWA-estimated out-of-school SB reached agreement when the equivalence zone was set at 15% .−1 (in-school MVPA) to 47.2 (91.5) min·week−1 for SB in primary schools, and from −4.5 (28.8) min·week−1 (weekend MVPA) to 118.4 (50.0) min·week−1 (SB) in secondary schools. Primary school YAP-predicted and SWA-estimated in-school and weekend MVPA demonstrated the closest agreement . For secondary school students, weekend MVPA and SB were equivalent in the 15% zone. Between-segment differences in bias were similar for boys and girls. These ranged from −2.5 (51.6) min·week−1 (boys) and −3.9 (71.1 min·week−1 (girls) for out-of-school MVPA, to 89.7 (78.2) min·week−1 (boys) and 61.2 (84.1 min·week−1 (girls) for out-of-school SB. Boys’ YAP-predicted and SWA-estimated out-of-school MVPA demonstrated the closest agreement . For girls, in-school and out-of-school MVPA were equivalent in the 15% zone. These additional school-level analyses are also presented in As the final calibration algorithms were specific to school-level and sex, additional sub-group agreement analyses were performed separately for all primary and secondary schools , and for−1 MVPA was achieved by 81% of the participants. YAP-predicted estimates of daily MVPA indicated that the recommendation was met by 85.8% of participants. Agreement was 80.7% and the kappa value was 0.31 (fair agreement). Sensitivity and specificity were 91% and 37%, respectively. The school day 30 min·day−1 MVPA recommendation was achieved by 77.6% and 79.2% of participants, according to SWA and YAP-predicted estimates, respectively. Percent agreement and kappa values were 82.2% and 0.47, respectively (moderate agreement). The classification accuracy was 89% sensitivity and 57% specificity.According to the SWA, 60 min·dayThis study aimed to examine the predictive accuracy of the US YAP algorithms for MVPA and SB in a sample of English youth, and to calibrate and test the validity and predictive utility of new English YAP algorithms. We found that the US YAP algorithms poorly predicted SWA estimates of MVPA and SB in English youth. School-level predictions of in-school, out-of-school, and weekend MVPA, and out-of-school SB from the English YAP algorithms were promising, and the YAP demonstrated potential as a surveillance tool to identify prevalence of compliance to youth PA guidelines.There was poor student- and school-level agreement between the US YAP estimates of MVPA and SB and estimates from the SWA. The recommendation of Saint-Maurice and Welk that the US YAP algorithms be tested and refined on independent samples  is thereWhen the estimates from the English YAP algorithms were compared to those from the SWA, student-level agreement was poor. This is consistent with what has been observed previously for the YAP ,25, for −1 was much higher than the −3.0 min·week−1 [−1 [−1 of the SWA estimates, which is substantially less than the −21.7 min·week−1 and −17.8 min·week−1 observed by Saint-Maurice and Welk [School-level bias between English YAP-predicted MVPA and SB and estimates from the SWA were substantially better than those from the student-level analyses. The degree of bias between in-school YAP and SWA MVPA was comparable with previous YAP validations ,25. Furtn·week−1  and 3.4 ek−1 [−1  in theirand Welk , respectand Welk ). Converand Welk ,25.−1, which may reflect that engagement in SBs, like TV viewing, or gaming occurred sporadically rather than as part of a set structure and routine [A key difference between the English and US YAP estimates was over-prediction of in-school and out-of-school MVPA, and SB. The English YAP algorithms were based on the specific school schedules and daily routines of the participants in the calibration sample, although the same data processing methods as the previous YAP studies were used ,25. It i routine . As a re routine ,59.Agreement between YAP-predicted and SWA-estimated MVPA was greatest for the weekend YAP segment. Weekends often involve greater choices and time for recreational activities, but weekend schedules can also reflect regularly occurring activities such as household chores, caring for siblings, and sports practices, etc. Although not as structured as the school day, weekends can represent familiar and routinised contexts for some youth. Moreover, the YAP uses two of the 15 questions to ask about PA during whole weekend days. Longer recall periods  are hypothesised to inhibit accurate recall of PA behaviours . It is, −1 recommendations indicated that 91% and 89% of youth who achieved the respective recommended daily MVPA would be correctly identified as ‘active’ based on the English YAP algorithm predicted MVPA. These sensitivity values are superior to those reported in the original US YAP calibration study [−1) and 61% (30 min MVPA·school-day−1) [The English YAP algorithms demonstrated their utility to evaluate compliance with health-related PA recommendations. Sensitivity values associated with the 60 and 30 min MVPA·dayon study  and reinl-day−1) , compareThe calibrated YAP estimates of MVPA and SB have great potential utility for future research and PA promotion, as existing calibrated self-report instruments for English youth are not available. Strengths of the study included (1) the use of a proven, rigorous YAP protocol and methodology; (2) the use of manageable group sizes for data collection, which allowed the use of recall probes to enhance the participants’ recall accuracy; (3) the recording of detailed timetable information from each school to accurately determine each participant’s schedule during the week when they wore the SWA, so as to enhance the degree of temporal precision required for the calibration analyses; (4) the use of an independent sample for the cross-validation analyses, and (5) the choice of the SWA as the device-based measure, which has previously demonstrated superior agreement with criterion measures of free-living energy expenditure than other research-grade and consumer activity monitors . There aPoor agreement was observed in MVPA and SB derived from the US YAP algorithms and SWA worn by the English sample. YAP algorithms developed using the English sample data resulted in MVPA and SB estimates that had promising school-level agreement with the lowest error observed for weekend MVPA and out-of-school SB. The YAP has potential as a surveillance tool to monitor compliance with youth PA guidelines, but more refinement is needed to improve its classification accuracy. The school-level YAP estimates of MVPA indicate that the YAP is a promising self-report questionnaire for use with English youth, and potentially with samples from other countries in the UK. The YAP is a cost-effective, easy to implement instrument that can be used at scale and implemented by researchers and practitioners to provide meaningful school-level estimates of MVPA and SB. Further refinement of the YAP algorithms with a more representative UK sample and by employing replicate measurement error modelling procedures to enhance the precision of the calibration algorithms is advocated.


Sp-CFSH1 and Sp-CFSH2) encoding the same CFSH precursor were cloned from the mud crab Scylla paramamosain. Homology and phylogenetic analysis showed that CFSHs were homologous to interleukin-17 and highly conserved among brachyuran crabs. PCR analysis revealed that Sp-CFSH was expressed exclusively in the eyestalk ganglion of both prepubertal males and females, and surprisingly, the abundance of Sp-CFSH transcripts detected in the males were not significantly different from that of the females (P > 0.05). In addition, mRNA in situ hybridization showed that Sp-CFSH was localized in the X-organ of the male eyestalk ganglion. During the development of the androgenic gland (AG), the level of Sp-IAG mRNA in AG remained at low levels from stages I to II (early stage) but had a significant increase at stage III (mature stage). In contrast, the level of Sp-CFSH transcripts in the eyestalk ganglion was high in the early stage but extremely low in the mature stage. To investigate the potential function of CFSH in male S. paramamosain, the recombinant protein (∼20 kDa) was expressed in Escherichia coli and was subsequently added to AG explants in vitro. It was demonstrated that recombinant Sp-CFSH protein significantly reduced the expression of Sp-IAG in the AG explants at a concentration of 10−6 M (P < 0.05). In conclusion, our study provides the first piece of evidence that shows CFSH from the eyestalk ganglion acts as a negative regulator inhibiting the development of AG in crustaceans.Recently, the crustacean female sex hormone (CFSH), which is considered a female-specific hormone, has been shown to play a crucial role in female phenotypes in crustaceans. In this study, two transcripts ( Eriocheir japonicus, the first pair of pleopods in males starts to appear at the third juvenile crab stage, but the third to fifth pleopods degenerate in males, while the second to fifth pleopods continue to develop and branch into two podites in females at the fourth juvenile crab stage , an organ unique to males . The funmarellus . Thereaf vulgare , 1999. B vulgare . For insreversal . Furtherogenesis . It has lt males .Callinectes sapidus) and demonstrated that it played a crucial role in the development of female reproductive phenotypes. It was shown that the silencing of CFSH or bilateral eyestalk ablation impaired the mating and maternal care structures of females, such as absent or misplaced gonopores, sharper abdomens, as well as shorter and fewer setae on pleopods  from the prokaryotic expression system and subsequently added it into AG explants to vindicate the role of CFSH in inhibiting Sp-IAG expression.In this study, we investigated a putative function of CFSH in male mud crab S. paramamosain) used for the experiments were obtained from a local fish market in Xiamen, Fujian, China. They were maintained in rectangular tanks with seawater at a temperature of 27 ± 2°C and a salinity of 28 ± 0.5 ppt for a week, during which they were fed with the clam Ruditapes philippinarum. Before tissue dissection, prepubertal crabs  at intermolt stage were placed on ice for anesthetization (n = 6). Various tissues including eyestalk ganglion, thoracic ganglion, cerebral ganglion, hepatopancreas, hemocytes, muscle, heart, stomach, gill, epidermis, Y-organ, mandibular organ, and gonad (ovary or testis) were then collected for tissue distribution analysis.The mad crabs (S. paramamosain can be classified into four stages (n = 6) at stage I , stage II , and stage III  were collected for Sp-CFSH and Sp-IAG expression (GenBank accession number: KJ870255) profiles analysis. Tissues were immediately frozen in liquid nitrogen, and stored at −80°C until total RNA extraction.The development of AG in r stages : At stag® reagent (Invitrogen). The first-strand cDNA was generated with 1 μg total RNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas). Degenerate primers CFSH-DF1 and CFSH-DR1 were designed based on the CFSH sequences of C. sapidus (GenBank accession number: GU016328.1) and Carcinus maenas (GenBank accession number: HM594946.1). The PCR reaction was performed in the ABI 2720 Thermal Cycler (Applied Biosystems) with Ex-Taq polymerase (Takara) under the following conditions: 95°C for 3 min; 35 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 50 s, followed by 72°C for 10 min final extension. After the amplification, the products were visually examined by 1.0% agarose gel. Subsequently, the fragment of Sp-CFSH was purified and then inserted into PMD19-T vector (Takara) for sequencing.Total RNA was extracted from the eyestalk ganglion of a mature female via TRIzolSp-CFSH, 5′ and 3′ rapid amplification cDNA ends (RACE) were performed with SMARTTM RACE cDNA Amplification Kit (Clontech) according to the manufacturer’s instructions. The 5′-RACE cDNA template was first amplified with primers CFSH5R1 and the universal primer, and then a nested PCR was performed with the dilute amplicon by primer CFSH5R2 and the short universal primer in the same conditions. The 3′-ends of the sequences were obtained by using 3′-RACE cDNA with universal primer and CFSH3F1, and nested amplification with nested universal primer and CFSH3F2. The two rounds of 3′-RACE-PCR were performed following the conditions of 5′-RACE-PCR. All PCR products were extracted using a 1.0% agarose gel and then subjected to sequence as described above. Finally, the sequences were assembled and its homology was examined using the Blast N. The primers were all listed in Supplementary Table To obtain the full length cDNA of 1. The signal peptide was predicted by Signal P4.02. The deduced CFSH amino acid sequences of crustacean species were arranged using Clustal X software. The phylogenetic tree was constructed by the maximum likelihood method with MEGA6 software.The open reading frame (ORF) was predicted by the ORF finderArginine kinase , a housekeeping gene, was used as an internal control. The PCR was carried out with primer pairs CFSH-QF/-QR and AK-QF/-QR . After being treated with DNase I (Fermentas) at 37°C for 30 min, 1 μg total RNA was reverse-transcribed with primer oligo-dT using a RevertAid First Strand cDNA Synthesis Kit (Fermentas). Tissues expression profiles were examined with a semi-quantitative PCR assay. in situ hybridization, a pair of gene-specific primer, CFSH-IF and CFSH-IR  E. coli by transformation for protein expression. Transformed cells were cultured at 37°C in LB medium with 50 μg/ml ampicillin. When OD600 reached to 0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.8 mM and cells were cultured for another 8 h at 18°C for protein expression. After harvest by centrifugation, cell pellets were resuspended in binding buffer  and dissociated by ultrasonic wave. The soluble fraction and insoluble fraction were separated by centrifugation at 12,000 × g for 15 min at 4°C and analyzed by 15% SDS–PAGE. The precipitate was harvested and resuspended in binding buffer containing 8 M urea, loaded on an Ni-NTA HisTrapTM FF crude column  according to the manufacturer’s instructions, and then washed with 10 ml of the elution buffer  containing 50 mM imidazole, 10 ml of the elution buffer containing 100 mM imidazole, and 5 ml of the elution buffer containing 200 mM imidazole in order. The rCFSH was eluted with 6 ml of the elution buffer containing 300 mM imidazole. Eluting solution were collected and examined by 15% SDS–PAGE. After that, purified rCFSH was renatured using the measure of graded urea dialysis and confirmed by Western blotting. For immunoblotting analysis, the electrophoresed proteins were transferred to a PVDF membrane and analyzed as previously described by The fragment encoding the mature hormone of in vitro experiment. The male crabs  were anesthetized on ice for 10 min and sterilized in 75% ethanol for 10 min. AG which is attached to spermaduct were immediately dissected and washed gently by crab saline with antibiotics penicillin G (300 IU/ml) and streptomycin (300 mg/ml) for five times. The AGs were placed in a 24-well cultured plate with 400 μl L15 medium containing antibiotics right after being separated from spermaduct and pre-cultured for an hour. The experiment was divided into two groups: with rCFSH at a final concentration of 10−6 M as experimental group and without rCFSH as control group, each group has four repeats (n = 4). Finally, AG samples were incubated for another 12 h and RNA was isolated for qRT-PCR assay.The biological activity of rCFSH for inhibiting AG was determined by an Sp-CFSH in the eyestalk ganglion and Sp-IAG in the AG, respectively. The cDNA samples were generated using TransScript® II One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech) according to the manufacturer’s protocol. 1 μg total RNA was reverse-transcribed with random primer and the cDNAs generated were diluted four times for qRT-PCR analysis. The reaction system of the qRT-PCR in this study was 20 μl in volume containing 10 μl 2× PCR Master Mix with SYBR Green, 2 μl dilute cDNA, 0.8 μl each primer (1 mM), and 6.5 μl water. The reaction was performed with 7500 Fast Real-Time PCR (Applied Biosystems) under the following conditions: 94°C for 10 min followed by 40 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, and a final extension 72°C for 10 min. The reactions were completed in triplicate and normalized to the internal control gene AK. Primers used for qRT-PCR were all listed in Supplementary Table The qRT-PCR was used to determine the relative expression of −ΔΔCt method, and then performed statistical analysis. Levene’s test was used for homoscedasticity and statistical significance of the data (P < 0.05) was determined using one-way analysis of variance (ANOVA) with post Duncan multiple comparison test (SPSS 20.0). The results were presented as mean ± SEM.We calculated the qRT-PCR data using the 2CFSH, named Sp-CFSH1 (GenBank accession number: MF489232) and Sp-CFSH2 (GenBank accession number: MF489233), were cloned from the eyestalk ganglia of S. paramamosain by using PCR with degenerate primers, followed by 5′- and 3′-RACE. It was found that the nucleotide sequences of Sp-CFSH1 (1032 bp) and Sp-CFSH2 (916 bp) were identical in 5′-UTR and ORF but different in 3′-UTR , which could be translated into a same hormone (223-aa). The predicted CFSH contained a 23-aa signal peptide, a 33-aa CFSH precursor-related peptide, a dibasic cleavage site (KR), and a 167-aa mature CFSH  in order, suggesting that it was synthesized as a preprohormone. There were eight cysteine residues that formed four putative intramolecular disulfide bridges in the mature CFSH: C46-150, C80-112, C105-119, and C107-148 .Two transcripts of Sp-CFSH share 95 and 90% identity with that of CFSH in C. sapidus and C. maenas, respectively. Phylogenetic analysis revealed that CFSH is a highly conserved hormone among brachyuran crabs . The mature hormones of CFSH, as well as CFSH-like, were determined to contain an interleukin-17 (IL-17) domain, indicating that they belonged to a new protein family homologous to the IL-17 family in vertebrates . In decapod crustaceans, CFSH can be classified into two types .The amino acid sequences of Sp-CFSH expression analysis were from prepubertal females and males. This revealed that Sp-CFSH was exclusively expressed in the eyestalk ganglia of both sexes of S. paramamosain . Unexpectedly, Sp-CFSH was found to be highly expressed in the eyestalk ganglia of males, which was not significantly different from that of females (P > 0.05) .Tissue distribution was determined by PCR with sequence specific primer pairs. The cDNAs of various tissues used for Figure 5E). The X-organ is located at the MT . The antisense Sp-CFSH probers gave a clear signal in cytoplasm of neuroendocrine cells in the X-organ of male . No signal was observed when a sense probe was used as a negative control .The eyestalk ganglia can be divided into lamina ganglionaris (LG), medulla externa (ME), medulla interna (MI), and medulla terminalis (MT)  of AG development . On the contrary, the level of Sp-IAG transcript in the AG increased during the development of AG and reached the peak at stage III, almost 40-fold higher than the level detected at stage I (P < 0.001) . This result was in accordance with the histological results that showed the volume of AG, as well as the number of endocrine cells, increased substantially at stage III compared with stage I and stage II .The expression profiles of elopment , Sp-CFSHE. coli BL21 with recombinant plasmid, induced with IPTG, in addition to the soluble and insoluble fractions were analyzed by 15% SDS–PAGE  was expressed after IPTG induction for 8 h. In contrast to the supernatant, the fusion protein was mostly expressed in the precipitate of ultrasonically broken E. coli BL21 (DE3). High-purity protein products were obtained after using Ni-NTA column affinity chromatography at 300 mM imidazole concentration  .Using the level of C. sapidus is a major breakthrough . Sp-CFSH1 (1032 bp) was longer than Sp-CFSH2 (916 bp) and the identified CFSH in C. sapidus (927 bp) . It has been reported that microsatellites in the 3′-UTRs can cause transcription slippage and influence the stability of mRNAs  . The dif(927 bp) . Alternahinensis . Microsaof mRNAs . Howeverof mRNAs . It woulE. sinensis and three CFSH-like proteins were found in coexistence in P. clarkii, M. rosenbergii, and L. vannamei, respectively . It appears common that this gene have several paralogs in crustacean species , a result in agreement with the findings of an immunohistology study in C. sapidus by Libinia emarginata . In S. verreauxi, CFSH-like was also found to be expressed in both male and female eyestalk ganglia at roughly same levels by comparative transcriptome analysis . In fact, the low expression in mature males, in our experiment, was similar to that of the adult male C. sapidus ; correspondingly, the level of Sp-IAG transcripts rose sharply at stage III (P < 0.001) .AG and/or IAG have been shown to play a crucial role in regulating male sex differentiation. For example, in f Mr-IAG , and thent of AG . In addint of AG , and theeristics . In the Macrobrachium nipponense, it was reported that both gonad-inhibiting hormone (GIH) and molt-inhibiting hormone (MIH) had the inhibitory effects on IAG gene expression  led to the up-regulation of Sp-IAG expression . In this study, Sp-CFSH was found to have the opposite expression tendency compared to Sp-IAG during the development of AG. That is, at the early stage, high expression of Sp-CFSH transcripts was detected in eyestalk ganglion  whereas the expression of Sp-IAG transcripts was low in AG . Therefore, we hypothesized that CFSH might suppress the development of AG at this early stage. To verify this, the recombinant CFSH (rCFSH) was obtained to assay its bioactivity in vitro and the result showed that the Sp-IAG expression transcripts in AG were reduced by about 60% by rCFSH, at a concentration of 10-6 M . Such a result demonstrates that CFSH generates an inhibitory effect on AG activity. Hence, our results suggest that CFSH might regulate male sex differentiation via AG.In crustacean males, it is well known that AG is under the inhibitory control of the eyestalk ganglion, because eyestalk ablation can result in hypertrophy . The AG pression . In S. ppression , suggestS. paramamosain, the expression of Sp-CFSH was extremely low , while the level of Sp-IAG transcript was high . A possible explanation for this phenomenon could be that AG/IAG affected the expression of Sp-CFSH in eyestalk ganglion in reverse. In the red claw crayfish Cherax quadricarinatus, implantation of AG into immature females caused the inhibition of female secondary sex characteristics  of male eristics , especiaeristics ; it is neristics . ClearlySp-CFSH from the eyestalk ganglion of the mud crab S. paramamosain. It appears for the first time that CFSH has been reported to be highly expressed in male crustaceans; and the inhibitory effect of CFSH on AG has been demonstrated. Furthermore, our research suggests the existence of putative antagonism between CFSH and IAG in male S. paramamosain, which warrants future investigation.In summary, our study characterized two transcripts of in situ, and statistical analysis. YH was involved in histological analysis, qRT-PCR analysis, data analysis, and GW was involved in the data analysis and preparation of the manuscript. HY designed the experiments, analyzed the results, and prepared the manuscript.AL carried out the majority of the experiments and prepared the manuscript. JL and FL were involved in cDNA cloning, hybridization The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Litopenaeus vannamei IRF (LvIRF) gene that has been shown to mediate antiviral responses by inducing the expression of Vago, a functional homolog of mammalian IFN. We then explored the effects of varying the number of (CT)n repeats on the expression of LvIRF using both dual-luciferase reporter assays and Western blots. Our results demonstrate that the length of the (CT)n-SSR in this gene can influence the expressional level of LvIRF, in that a shorter (CT)n repeat had a stronger ability to induce the expression of LvIRF. Moreover, we found that the (CT)n repeat in LvIRF was associated with viral resistance in shrimp. Individual shrimps with shorter (CT)n repeats in the 5′-UTR of LvIRF exhibited high tolerance to white spot syndrome virus (WSSV), and this trait was inherited in offspring. Taken together, these results indicated that this (CT)n-SSR could be used as a molecular marker for shrimp breeding for WSSV resistance.Simple sequence repeats (SSRs) of short nucleotide motifs occur very frequently in the 5′ untranslated coding region (5′-UTR) of genes and have been implicated in the regulation of gene expression. In this study, we identified an SSR with a variable number of CT repeats in the 5′-UTR of the  Simple sequence repeats (SSRs), also known as microsatellites or short tandem repeats (STRs), are tandem repeats of short sequence motifs occurring ubiquitously in eukaryotic genomes. They exhibit extensive polymorphism due to variations in the copy number of each specific repeat motif, and are considered useful as genetic markers for genetic diversity analysis, DNA fingerprinting, and linkage mapping. Several studies have suggested that SSRs are non-randomly distributed in the genome, as untranslated coding regions (UTRs), and have more SSRs than coding regions, as well as exhibit a strong bias towards di- and tri-nucleotides repeats . SSRs inFMR1) gene expression due to a long CGG tri-nucleotide repeat in the FMR1 gene 5′-UTR adjacent to the promoter that results in the FMR1 gene’s epigenetic silencing (n microsatellite identified in the 5′-UTR of the prolactin 1 (prl 1) gene of tilapia has been associated with differences in prl 1 gene expression and the growth responses of salt-challenged tilapia n microsatellite sequences in promoter regions have been shown to bind to a protein family called GAGA factors, which are involved in gene expression  has been attributed to the absence of fragile X mental retardation 1  (The shrimp species s (WSSV) . To dateL. vannamei IRF (LvIRF) is the first interferon regulatory factor (IRF) identified in crustaceans, and has a similar protein nature as mammalian IRFs. LvIRF can be activated during viral infection, and then translocates to the nucleus to initiate the expression of the L. vannamei Vago4 (LvVago4) gene. This is effectively an arthropod cytokine encoding a viral-activated secreted peptide through activating the JAK-STAT pathway to restrict viral infection, similar to mammalian IFNs n repeats modulates the promoter activity of the LvIRF gene in a length dependent manner, and observed that shrimp with different numbers of CT repeats showed distinct tolerances to WSSV. Furthermore, we demonstrate the use of the number of (CT)n repeats at this locus as a molecular marker to selectively breed a new generation of shrimp resistant to WSSV. In F2 offspring, the populations with smaller numbers of (CT)n repeats were more resistant to WSSV.The key constituents of innate immunity were regarded as molecular markers for breeding disease resistance in shrimp. In this study, we found a (CT)n repeats and the antiviral traits of L. vannamei, three different populations of 100 healthy shrimp  were collected from the Hengxing shrimp farm in Zhanjiang city, China. TH01 was collected from Vietnam, TH03 was collected from China, and SP07 was collected from Saipan. The shrimp body weight was 5.0 ± 1.0 g each, and each population of shrimp was cultured in filtered sea water with a 2.5% salinity at 26°C in a recirculating water tank and fed with fodder at rate of 5% of body weight per day. WSSV was prepared from shrimp muscle tissue previously infected with WSSV and stored at −80°C. The muscle tissue was homogenized to prepare it as a WSSV inoculum at a final concentration of 106 virions/50 μl, which was injected into each shrimp (th day (240 h) after infection, all shrimp were collected and marked. In a study of the WSSV resistance of L. vannamei, the survival rate curve showed a normal distribution using this injection infection method (In order to investigate the relationship between (CT)h shrimp . All tann method . TherefoLvIRF cDNA had previously been deposited in the NCBI GenBank (KM277954), and the L. vannamei IRF-5′-UTR was used as a DNA template for PCR amplification . The fication . PCR profication , and thoLvIRF without a stop codon was used as a cDNA template for PCR amplification (LvIRF-5′-UTR sequences were then amplified from pMD-19T vectors and cloned into the pAc-IRF-V5 vector to generate pAc-IRF-(CT)n-V5. These were sequenced, and a pair wise alignment was then performed. The promoter regions of LvVago4 were cloned into the PGL-3 basic vector  to generate a luciferase reporter gene plasmid  and reverse transcribed into cDNA using the PrimeScript RT reagent kit . The ORF of  plasmid .LvIRF on the promoters of LvVago4 genes, dual-luciferase reporter assays were performed using Drosophila Schneider 2 (S2) cells with pAc-IRF-(CT)n-V5 that contained different numbers of CT repeats as IRF-expressing vectors. Cells in each well of a 96-well plate  were transfected with 0.05 μg PGL-LvVago4 vector as a reporter plasmid, 0.005 μg pRL-TK renilla luciferase , and 0.05 μg of expression plasmid, LvIRF vector or empty expression vector as controls. The pRL-TK renilla luciferase plasmid was used as an internal control. 48 h after transfection the firefly and renilla luciferase activities were measured, according to the manufacturer’s instructions. Each experiment was performed in triplicate.To detect the effects of n-V5 vector was transfected into S2 cells. After 72 h, cells were harvested and lysed in NP-40 lysis buffer with protease inhibitor cocktail . Western blot was performed with a rabbit anti-V5 antibody  as a primary antibody, and alkaline phosphatase-conjugated goat anti-rabbit as a secondary antibody . ImageJ was then used to measure the gray-level values and calculate the ratio of LvIRF and β-actin. Quantitative RT-PCR was performed for the analysis of IRF gene expression, and the EF-1α gene was detected as an internal control (The pAc-IRF-(CT)LvIRF 5′-UTR region was genotyped using FAM fluorogenic probes followed by capillary electrophoresis to discriminate allele size. Primers (n repeat numbers ranged from 13 to 24) that was adopted by referring to other reports  and 95% confidence intervals (95% CIs) to calculate significance, as well as two-tailed unpaired t-tests and ANOVA. Bonferroni corrections were applied to account for the multiple testing of the CT genotype frequency [*p corr < 0.0042 (0.05/12)] and genotype groups [*p corr < 0.0167 (0.05/3)], following Fisher exact tests (post hoc tests (*p < 0.05).Allelic frequency calculations were performed using PopGen32 software . We usedct tests . ANOVA tLvVago4 to regulate its transcription, which plays a role in the defense against WSSV infection (n repeat in the 5′-UTR region of the LvIRF gene and designed primers to amplify the 5′-UTR including the (CT)n motif. PCR amplification of genomic DNA from 20 shrimp collected from diverse genealogies was performed and the amplified products had various lengths, ranging in size from 190 to 230 bp n repeat and a short (CT)n repeat. Fourteen sequences, different in (CT)n motif number only, were chosen for further study. A pair wise alignment was then made using part of these sequences, such as (CT)10+5, (CT)14+5, and (CT)18+7 n motifs were cloned into pAc-5.1-His/V5 A to generate pAc-IRF-(CT)n-V5 vectors for protein expression n motif number variation on LvIRF expression, Western blotting was then performed. The results showed that the ratio of LvIRF and β-actin with (CT)15 was twice as large as that with (CT)25 in terms of gray-level values (LvVago4 contains a conserved IRF binding motif that was confirmed to be targeted by LvIRF (LvVago4 promoter (LvIRF was observed in cells transfected with pAc-IRF-(CT)15, which was 1.5-fold higher than in cells with pAc-IRF-(CT)25 (p < 0.001), and 2.3-fold higher than in cells with pAc-IRF-V5 (p < 0.001). Considering that the (CT)n repeat is composed of two parts, we explored whether the different numbers of the two (CT)n repeats were implicated in LvIRF expression. We observed that the pAc-IRF-(CT)11+5 and pAc-IRF-(CT)10+6 showed no significant differences on the promoter activities of LvVago4. Similar results were observed in three other groups that contained a total of 17, 18, and 19 repeats, respectively n copies.To investigate whether the variable numbers of (CT)by LvIRF . To furtn repeats in TH01 were skewed towards 18, 19, or 20    , although this difference was only nominally significant n repeat numbers ranged from 13 to 24 in the three populations, the repeat length cut-offs for allelic categorization were defined as ≤18 repeats for short (S) alleles and >18 repeats for long (L) alleles. Following allele classification, we could attribute to each individual one of three bi-allelic genotypes: S/S, S/L, or L/L. Genotype distribution frequencies were then determined and are shown for each population in p < 0.0003, OR = 28.053, 95% CI = 3.417 - 230.33) and the L/L genotype . Similar to the TH01 population, the S/S genotype associated with WSSV resistance showed a significant difference in the TH03 population , while the L/L genotype showed a significant difference in the SP07 population .Since the (CT)LvIRF gene were resistant to WSSV, while those with L/L were susceptible to WSSV. The above results suggested that the shrimp with a short (CT)n repeat in the 5′-UTR of LvIRF were more resistant to WSSV than those with longer (CT)n repeats.Allelic distribution showed that S alleles tended to be enriched in Res. groups and L alleles tended to Sus. groups. We then compared the bi-allelic genotype distribution between the Sus. and Res. groups, and shrimp with the S/S genotype in the 5′-UTR of the n repeats in the 5′-UTR of LvIRF, and HX-CTL, which only contained long (CT)n repeats. 14 and (CT)15 genotypes, while the HX-CTL population did not. The survival rate curve after WSSV infection is shown in LvIRF gene expression in gills by quantitative RT-PCR . These results suggested that shrimp containing a smaller number of (CT)n repeats in the 5′-UTR of LvIRF were more resistant to WSSV infection, thereby improving their survival rate in this assay.Based on our breeding scheme illustrated in n motif in the 5′-UTR, as well as its repeat length variation, in the transcriptional regulation of the IRF gene of L. vannamei and the association of its genetic variations with WSSV resistance. Microsatellite repeats, especially (CT)n motifs, exist in abundance in the 5′-UTRs of several eukaryotic genes n variation in the 5’-UTR of the LvIRF gene, we selected (CT)n variant alleles from different individuals and found that shrimp with smaller numbers of (CT)n repeats exhibited an enhanced tolerance to WSSV infection.Our study examined the role of an SSR with a (CT)ic genes . The micic genes . Our firCatharanthus roseus, Tryptophan decarboxylase (Tdc) gene expression is affected by the length of a (CT)n microsatellite in the 5’-UTR of this gene n repeat motifs present in the 5’-UTR, were differentially transcribed, and were shown to be associated with drought tolerance n repeat numbers. In particular, shorter repeats [(CT)15] showed higher expression of LvIRF than longer repeats [(CT)25].SSRs in the UTRs sequences or within promoter regions are thought to play a role in the regulation of gene expression. In his gene . The chiolerance . Recentlolerance . In our LvIRF is an IRF in crustaceans, with functions similar to mammalian IRFs. LvIRF mediates the IRF-Vago-JAK/STAT pathway in shrimp, and has been shown to inhibit viral (WSSV) replication, analogous to the IRF-IFN-JAK/STAT pathway in vertebrates (LvIRF genotype and WSSV resistance. Our results demonstrated that the LvIRF (CT)n repeat number was associated with WSSV resistance in shrimp. Smaller numbers of repeats showed significant resistance to WSSV, perhaps due to the higher LvIRF expression. We selected three populations of shrimp with different genetic backgrounds and allelic distributions of LvIRF to analyze the association between (CT)n repeats and WSSV resistance, and obtained similar results. This indicated that the regulation of LvIRF expression by an SSR with a (CT)n motif in the 5′-UTR was widespread in L. vannamei. Studies have shown that the IRF-IFN-JAK/STAT pathway is a broad-spectrum antiviral pathway in vertebrates n repeats had a stronger antiviral immune response, as manifested by observation of an increased LvIRF gene expression and the prolonged survival time after WSSV infection. Thus, the identified SSR could be candidate marker for shrimp breeding for WSSV resistance. In fact, molecular markers have been widely reported in the breeding of several cash crops, including rice and wheat n -SSRs could influence the levels of gene expression. In addition, we also demonstrated that this SSR was associated with WSSV resistance. Our results provide some insights into how this SSR could be used as a molecular marker in breeding of WSSV-resistant shrimp. Further work needs to be done to investigate the feasibility of other molecular markers in shrimp breeding.In summary, we identified an SSR of a (CT)All reagents and experimental data are available within the article or supplementary information or from corresponding author upon reasonable request.CL and JH conceived and designed the experiments. BY, HW, PZ, and SW performed the experiments and analyzed data. CL and BY wrote the draft manuscript. CL was responsible for forming the hypothesis, project development, data coordination, and writing, finalizing, and submitting the manuscript. All authors discussed the results and approved the final version.This research was supported by National Key Research and Development Program of China (2018YFD0900502/5), the Science and Technology Planning Project of Guangdong Province (2018B020204001); National Natural Science Foundation of China (31772883); Guangdong Natural Science Funds for Distinguished Young Scholars (2016A030306041); Tip-Top Scientific and Technical Innovative Youth Talents of Guangdong Special Support Program (No. 2016TQ03N504), and Fundamental Research Funds for the Central Universities (17lgpy62). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer JX declared a shared affiliation, with no collaboration, with the authors to the handling editor at time of review.


Deep learning approaches have achieved state-of-the-art performance in cardiac magnetic resonance (CMR) image segmentation. However, most approaches have focused on learning image intensity features for segmentation, whereas the incorporation of anatomical shape priors has received less attention. In this paper, we combine a multi-task deep learning approach with atlas propagation to develop a shape-refined bi-ventricular segmentation pipeline for short-axis CMR volumetric images. The pipeline first employs a fully convolutional network (FCN) that learns segmentation and landmark localisation tasks simultaneously. The architecture of the proposed FCN uses a 2.5D representation, thus combining the computational advantage of 2D FCNs networks and the capability of addressing 3D spatial consistency without compromising segmentation accuracy. Moreover, a refinement step is designed to explicitly impose shape prior knowledge and improve segmentation quality. This step is effective for overcoming image artefacts (e.g. due to different breath-hold positions and large slice thickness), which preclude the creation of anatomically meaningful 3D cardiac shapes. The pipeline is fully automated, due to network’s ability to infer landmarks, which are then used downstream in the pipeline to initialise atlas propagation. We validate the pipeline on 1831 healthy subjects and 649 subjects with pulmonary hypertension. Extensive numerical experiments on the two datasets demonstrate that our proposed method is robust and capable of producing accurate, high-resolution and anatomically smooth bi-ventricular 3D models, despite the presence of artefacts in input CMR volumes. ICARDIAC magnetic resonance (CMR) imaging is the gold standard for assessing cardiac chamber volume and mass for a wide range of cardiovascular diseases . For decTheoretically, 3D neural network-based segmentation methods may be designed with arbitrarily deep architectures. In practice however, the size of cardiac images, especially that of high-resolution volumetric images , often pDue to the limitations of standard clinical acquisition protocols, raw volumetric CMR images acquired from standard scans often contain several artefacts , includi1 , the 2011 LV segmentation challenge2 (organized as part of the STACOM workshop), the 2015 RV segmentation challenge , , , 12], , , 14], ,  have beehallenge  and the We propose a multi-task deep learning network that simultaneously predicts segmentation labels and anatomical landmarks in CMR volumes. The network takes input volumetric images as multi-channel vector images (2.5D representation), requires no ROI extraction, and contains up to 15 convolutional layers. As such, the network has the computational advantage of 2D networks and is able to address 3D issues without compromising accuracy and spatial consistency. To our knowledge, this is the first work applying deep learning to CMR landmark localisation in a 3D context.We introduce anatomical shape prior knowledge to the network segmentation, which is a refinement step that is carried out using atlas propagation with a cohort of high-resolution atlases. As such, the pipeline is able to produce an accurate, smooth and clinically meaningful bi-ventricular segmentation model, despite the existing artefacts in the input volume. Moreover, due to the use of landmarks detected by the network, the proposed pipeline is entirely automatic.We demonstrate that the proposed pipeline can be readily generalised to segmenting volumetric CMR images from subjects with pulmonary hypertension (a cardiovascular disease). We thoroughly assess the effectiveness and robustness of the proposed pipeline using a large-scale dataset, comprising 2480 short-axis CMR volumetric images for training and testing. To our knowledge, this is one of the first CMR segmentation studies utilising a volumetric dataset of this size, and the technique introduced herein is the first automatic approach capable of producing a full high-resolution bi-ventricular model in 3D.In this paper, we propose a segmentation pipeline to address the aforementioned limitations of current approaches. Specifically, we make the following contributions:IIA3 view  and high-resolution (HR) volumes. 3 view b. MoreovThe proposed pipeline has three main components: segmentation, landmark localisation and atlas propagation. We term the proposed network used in the pipeline as the Simultaneous Segmentation and Landmark Localisation Network (SSLLN). Further, the related terms SSLLN-HR and SSLLN-LR will be used to refer to versions of SSLLN trained with HR and LR volumetric data, respectively. In BS = {, i = 1t, …, N}, where iU  stochastic gradient descent α and β are weight coefficients balancing the three terms. DL (W) is the segmentation loss that evaluates spatial overlap with ground-truth labels. LL(W) is the landmark associated loss for predicting landmark locations. W. This term is used to prevent over-fitting in the network. The training problem is to estimate the parameters W associated with all the convolutional layers and by minimising (DL (W) above is first given as follows {·} is an indicator function. ϵ is a small positive value used to avoid dividing by zero. i, k and j respectively denote the training sample index, the segmentation label index and the voxel index. j  represent background). To solve this highly imbalanced classification problem, we propose the class-balanced weighted categorical cross-entropy loss LL(W)=−∑iLL(W)=−∑i× [3D volume size] × classification categories) during forward and backward propagations and requires far more GPU memory than their 2D counterparts. Workarounds such as subsampling ,  range.Image preprocessing was carried out to ensure: 1) the size of each volumetric image fits the network architecture; 2) the intensity distribution of each volume was in a comparable range so that each input could be treated equally importantly. As such, each of the HR volumes in Dataset 1 was reshaped to common dimensions of 192 x-, y- or z-axis) at the central voxel of the volume. Neither shearing nor volume flipping was used. Data augmentation enables the network to see a large and diverse array of inputs by the end of training, and was implemented using the SimpleITK library in Python. With an Nvidia Titan XP GPU, training (50 epochs) took approximately 20 and 10 hours for Datasets 1 and 2, respectively. For inference, segmentation (without shape refinement) of an HR/LR volume for a single subject at ED took < 1s.Since our network takes volumetric images as inputs, we performed 3D data augmentation on-the-fly during training. At each iteration, augmentation included rescaling of voxel intensities in the input volume, and a 3D random affine transformation of the volume and corresponding labels and landmarks. For simplicity, the affine transformation only involved in-plane translation, isotropic scaling and rotation along one random direction . Note that the proposed SSLLN-HR is a multi-task network that simultaneously outputs labels and landmarks. Next we segmented a cardiac volume into 5 regions: the left ventricular cavity (LVC), right ventricular cavity (RVC), left ventricular wall (LVW), right ventricular wall (RVW) and background. Our method is the first one capable of producing a full HR bi-ventricular segmentation (LVC+LVW+RVC+RVW) in 3D.5 was trained using each short-axis slice in the volume separately. The second one is the 3D FCN, whose architecture is similar as in × 112 × 64. To focus exclusively on segmentation accuracy, we removed the landmark localisation activation maps in the last layer of the 3D FCN. As In k consecutive short-axis slices in a volumetric image, with multiple settings of k . In each case, we set intensities in the selected slices to zero, as shown in the 1st column. The two methods under comparison were then applied to these partially zero-filled volumes, and the results are given in 2nd-5th columns. As is evident, 2D FCN fails to segment these zero-filled slices, thus leaving gaps in the resulting 3D segmentations. In contrast, SSLLN-HR demonstrates robustness to missing slices and has the capability of ‘inpainting’ these gap regions. However, as the gap (number of zero-filled slices) increases (from k=5 to k=20), the segmentation performance becomes worse. These results further illustrate that the proposed network retains 3D spatial consistency, which the 2D FCN is unable to achieve. Our method thus outperforms the 2D approach in this regard.In DTo enable automatic alignment for subsequent non-rigid registration, we also predicted landmark locations (together with segmentation) for each input volume using SSLLN-HR. Same as above, we used the split subsets 1000/831 for training and testing. Note that SSLLN-HR was trained with manual landmarks carried out by inter-user 1 on each of the 1000 subjects. For the 831 unseen test subjects, the automatically detected landmarks were compared with the manual ones from inter-user 1 using the point-to-point Euclidean distance. Also, to study inter-user variability of landmarking, a second expert (inter-use 2) was recruited to manually annotate landmarks for each of 831 test subjects. The annotations were then compared with those of inter-user 1.±3.20 mm for Landmark-I to 8.18±6.91 mm for Landmark-II. In contrast, the errors between the two inter-users vary from 5.61±2.62 mm for Landmark-V to 17.4±9.27 mm for Landmark-II. This confirms that computer-human difference can be smaller than human-human difference.In EIRTK implementation6, it took ~0.005s to compute an affine transformation on a pair of landmarks, whilst it took ~5s to perform an affine registration using the 3D segmentation labels with size 256 × 256 × 56.In this section, we show that landmarks localisation is a necessary step in our pipeline. In We note that it may also be possible to detect landmarks automatically from segmentation labels. In this case, the accuracy of landmarks will be conditioned on the accuracy of segmentation. On the other hand, it may not be straightforward to determine which landmarks should be detected from segmentation labels for robust registration. As such, directly detecting the six landmarks defined in the study using the proposed network is neater and better.F×1.25×2 mm to 1.25×1.25×10 mm, as shown in the 1st and 2nd columns. The downsampling produces a staircase artefact due to reduction in long-axis resolution. Moreover, the segmentation (To quantitatively assess the performance of SSLLN-LR and shape refinement (SSLLN-LR+SR) in the pipeline (bottom path in entation d around n=231) were used to refine SSLLN-LR segmentations.Next, for training the SSLLN-LR, the LR volume Here we highlight three reasons why we used SSLLN-HR network results as a reference atlas set for shape-refinement: 1) our SSLLN-HR is able to produce results that are very similar to the corresponding ground truth, as confirmed from In In Table III7 (MAM)  ma ma8 coul


Owing to their natural abundance and exceptional mechanical properties, cellulose fibers (CFs) have been used for reinforcing polymers. Despite these merits, dispersing hydrophilic CFs in a hydrophobic polymer matrix is challenging. To address this, an amphiphilic ammonium salt was employed as the dispersant for CFs in this study. The hydrophobic CFs were mixed with a healable polymer to produce CF-reinforced composites. As the thermosetting polymer was crosslinked with Diels–Alder (DA) adducts, it was mended and recycled via a retro DA reaction at 120 °C. Interestingly, the CF-reinforced polymer composites were mended and recycled as well. When 5 wt % of the hydrophobic CFs was added to the polymer, maximum tensile strength, elongation at break, Young’s modulus, and toughness increased by 70%, 183%, 75%, and 420%, respectively. After recycling, the CF-reinforced composites still featured better mechanical properties than recycled polymer. As cell bonding . This pr bonding . Althougnding [4 , acid annding [4 , carbonynding [4 ,8, TEMPOnding [4 ,10, deepnding [4 ,12, and nding [4 . AlthougPolymers containing reversible bonds can mend damage when they are impacted, which ultimately extends their lifetime ,15. PolyMn ~ 1000 g/mol), tetramethylammonium chloride (TM), tetrabutylammonium chloride (TB), hexadecyltrimethylammonium chloride (TH), propylene glycol monomethyl ether acetate (PGMEA), methyl ethyl ketone (MEK), dimethylformamide (DMF), and dibutyltin dilaurate were purchased from Sigma-Aldrich Korea Ltd, Yongin, Korea. Isophorone diisocyanate (IPDI), bismethane (BMI), and furfurylamine were purchased from SEJINCI, Seoul, Korea. Cellulose was purchased from MOORIM P&P, Seoul, Korea. All chemicals were used as received, without purification. The furan diol 1 was synthesized by following the known method [Poly(propylene glycol)  was diluted with deionized water (100 g) to produce a CF suspension with a solid content of 1% by weight. This was mixed with hexadecyltrimethylammonium chloride (5.0 g) and stirred at room temperature. After 30 min, hydrophobic CFs were isolated by filtration using a Buchner funnel, and dried in an oven .1  and PPO-1000 . To this mixture were added IPDI  and PGMEA (16.6 g). A catalytic amount of dibutyltin dilaurate was added to the mixture at room temperature. The solution was heated at 60 °C for 3 h and cooled to room temperature to produce an F-PU solution with a solid content of 50% by weight.The flask was charged with furan diol To the as-prepared F-PU in PGMEA (5.0 g) was added BMI (0.33 g). This mixture was heated at 60 °C. After 20 min, the solution was poured into a PTFE mold, followed by being kept in an oven. Oven temperature was increased from 40 to 80 °C slowly to produce a DA-PU pad.To the as-prepared F-PU in PGMEA (5.0 g) were added the TH-treated CFs. Once the cellulose fibers were dispersed in the F-PU solution, BMI (0.33 g) was added and stirred at 60 °C for 20 min. The solution was poured into a PTFE mold, followed by being kept in an oven. Oven temperature was increased from 40 to 80 °C slowly to produce CF-reinforced composites. The ratio of CF/DA-PU ranged from 1% to 7% by weight.The as-prepared DA-PU solution was poured on a glass substrate with dimensions of 5 cm × 5 cm. Then, it was spun at 500 rpm for 20 s to form a thin polymer layer on the glass substrate. This was kept at 80 °C overnight to produce a DA-PU layer on the glass substrate. The DA-PU layer was scratched by a cutter knife, then it was kept at 120 °C for 20 min for healing. The change in the scratch before and after thermal treatment was monitored by optical microscopy. For the healing test of a composite, a solution containing CF-reinforced composite with a CF content of 5 wt % was used.d6 as a solvent at room temperature. FTIR spectra in the range from 600 to 4000 cm−1 were measured on a Fourier transform infrared spectrophotometer , by placing samples on the surface of a ZnSe crystal using the attenuated total reflectance method. Surface profiles were measured by laser confocal microscopy . The surfaces of the fractured samples were observed by scanning electron microscopy . Thermal stability of polymers, TH-treated CFs, and CF-reinforced composites were evaluated under N2 atmosphere by elevating the temperature from 23 to 400 °C at the rate of 10 °C/min using a differential thermal thermogravimetry analyzer . The molecular weight was obtained on an ACQUITY APC  equipped with a column (ACQUITY APC XT 2002.5 µm) by flowing a THF solution of a sample at the rate of 0.7 mL/min. After calibrating the column set using polystyrenes with molecular weights ranging from 1220 to 264,000 g/mol, the molecular weight of the sample was measured.The proton NMR spectra were measured at 400 MHz on an NMR spectrometer equipped with Bruker Top Spin 3.2 software  using DMSO-To make CFs hydrophobic, their aqueous solutions were treated with quaternary alkyl ammoniums (QAAs). The absorption of the QAAs on the surface of the CFs would alter the hydrophilicity of the CFs. We chose three types of QAAs  to investigate the effect of the alkyl groups on the hydrophobicity of the CFs. The changes in the hydrophobicity of the QAA-treated CFs were readily observed by the extraction method using a water/toluene mixture. In this, the aqueous suspension of CFs was diluted to 1% using de-ionized water and stirred for 30 min. The solution was stirred by a magnetic stirrer at RT for 30 min. Then, toluene was added to the CF suspension to make the ratio of water/toluene one-to-one by weight. After the two layers were mixed in a separatory funnel, the mixture was allowed to stand for 30 min to check whether the CFs had been transferred to the organic layer . The CFs1 was readily synthesized by reacting glycerol carbonate and furfuryl amine at the ratio of 1:1 following the reported method. Then, a mixture of furan diol 1, polyol (PPO-1000), and IPDI in PGMEA was heated at 60 °C for 3 h to produce F-PU, in which the furan groups were grafted onto the polyurethane backbones  was calculated to be 1.176.To fabricate healable composites using the TH-treated CFs, we synthesized polymers crosslinked by DA adducts. Furan diol ackbones . The comretching . The ideCOSY-NMR , in whic2 was added to F-PU in PGMEA, and the solution was spin-coated on a glass substrate followed by drying at 80 °C overnight. When the solvent was slowly evaporated, DA adducts formed to produce a free-standing film of DA-PU. Owing to the crosslinking through DA adducts, thermosetting DA-PU was not dissolved in organic solvents such as MEK and DMF at RT. To investigate a retro DA reaction, DA-PU was immersed in DMSO-d6 and kept at 120 °C for 20 min. DA-PU powder was dissolved in DMSO-d6 over time to make the solution yellow in color . The results showed that DA-PU was stable below 250 °C with little change in weight  and the elongation at break (88%) of the silicone-based polyurethane crosslinked with DA adducts in the previous report [Besides the self-healing ability, a retro DA reaction made the thermosetting polymers recyclable . To thisn weight . As thern weight . A polyms report .2 to both composite solutions, they were poured into PTFE molds and kept in an oven overnight. While the TH-treated CFs were well dispersed in the polymer, the pristine CFs aggregated in the matrix (Encouraged by this, we mixed TH-treated CFs and DA-PU to improve the mechanical properties of DA-PU. To compare the dispersibility of TH-treated CFs with that of pristine CFs, they were mixed with a PGMEA solution of F-PU, at the CF/F-PU ratio of 100-to-5 by weight, and sonicated for 30 min. After the addition of bismaleimde e matrix . This deThe stress-strain curves show that mechanical properties were improved as the CF content in the thermosetting composites increased . When thWe recycled the CF-reinforced thermosetting polymer composites by dissolving them in PGMEA at 120 °C, followed by pouring in a PTFE mold. The mechanical properties of the recycled ones were measured and are summarized in Hydrophobic CFs were not only dispersed in hydrophobic polymers but also acted as both fillers and plasticizers, which improved the mechanical properties of composites. The combined enhancement in Young’s modulus and elongation realized that toughness of the composites increased in proportion to the amount of the dispersed CFs compared to the unreinforced polymer. The incorporation of the hydrophobic CFs in thermosetting, crosslinked by DA adducts, resulted in highly-durable mendable composites without deteriorating healing ability. In addition, thermosetting composites were recyclable via a retro DA reaction, which is not possible for conventional thermosetting crosslinked by irreversible bonds.


Objective: To evaluated the prognostic ability of several serum cytokines in clinically isolated syndrome (CIS) patients regarding second events and conversion to multiple sclerosis (MS) or neuromyelitis optica spectrum disorder (NMOSD).Methods: We enrolled 69 CIS patients whose serum samples were collected during the acute phase of the first onset before immunotherapy. Fifteen other non-inflammatory neurological disorder (OND) patients were also included. The serum levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-13, IL-17A, IL-21, IL-23, interferon-γ (IFN-γ), and transforming growth factor beta 1 (TGF-β1) were measured using the human cytokine multiplex assay or ELISA. Patients were seen every 3–6 months. Unscheduled visits occur in case of exacerbations. Clinical measures of disease progression were recorded.Results: Twenty CIS cases had second events during follow-up at a mean time of 15.3 ± 9.9 months. Serum IL-10 levels were significantly lower in CIS patients who relapsed compared to patients who did not. Low serum IL-10 levels were associated with higher risk and shorter times to second events. In clinical correlations, a significantly higher CSF white blood cells count, number of T2 lesions, and gadolinium-enhancing (Gd+) lesions in baseline MRI were found in the low serum IL-10 level group. Of the 20 relapsed cases, seven converted to MS, and eight converted to NMOSD. No significant differences were found in any cytokine levels between these patients at first onset.Conclusions: These findings support using serum IL-10 as a biomarker associated with the risk of relapse and the time to second events in patients with CIS. However, serum cytokine levels can not differentiate between the conversion from CIS to MS or NMOSD. Idiopathic inflammatory-demyelinating diseases (IIDDs) are monophasic, multiphasic, or progressive disorders characterized by inflammatory demyelination affecting attacks different sites of the central nervous system (CNS) . This spClinically isolated syndrome (CIS) is a term widely used in contemporary neurological practice to describe the first clinical episode in which a patient has symptoms and signs suggestive of an inflammatory demyelinating disorder of the CNS . The terBetween January 2015 and August 2017, 228 consecutive patients hospitalized in the neurology department of Beijing Tiantan Hospital were diagnosed with CIS. Patients meeting the following conditions were included: (1) the serum samples were collected during the acute phase (< 2 weeks from symptom onset) of the first onset and before any immunomodulatory or immunosuppressive therapy, (2) any infectious or other autoimmune comorbidities did not coexist at the time of sample collection, and (3) patients did not receive immunomodulatory treatment before relapse, or during follow-up time for patients without relapse.Baseline clinical data including gender, age, routine CSF information , MRI information , and the Expanded Disability Status Scale (EDSS) disability score were recorded. Patients were seen every 3–6 months. Unscheduled visits occurred in case of exacerbations. The EDSS score during each clinic visit and the time to second event were prospectively collected. Relapses were defined as the development of new or recurrent neurological symptoms not associated with fever or infection lasting for at least 24 h. NMOSD and MS diagnoses were made according to the 2015 Revised International Criteria  and 2010n = 3), cluster headache (n = 3), psychogenic movement disorders (n = 3), normal pressure hydrocephalus (n = 2), benign paroxysmal positional vertigo (n = 2), sleep disturbance (n = 1), and vitamin B12 deficiency (n = 1).Fifteen patients with other non-inflammatory neurological diseases (OND) were also enrolled (13 women and two men with a mean age of 40.2 years). The OND group included benign intracranial hypertension , and written informed consent was obtained from all participants.The CSF samples were centrifuged and the supernatants were collected and stored at −80°C until analyzed. Samples were tested for the aquaporin-4 antibody (AQP4-Ab) using a cell-based assay (CBA) with live HEK-293 cells transiently transfected with full-length M23-AQP4, as described previously . The samp < 0.05.Statistical analysis was conducted using SPSS 22.0 . Data were presented as the mean ± SD. Continuous data were compared with the non-parametric Mann–Whitney test. Categorical data were compared with the chi-square or Fisher's exact test. Receiver operating characteristic (ROC) curve analyses were used to determine the best cut-off value based on serum IL-10 levels and CSF WBC count. Univariate and multivariable Cox proportional hazard regression models were used to evaluate the association between serum IL-10 levels and the time to relapse, including the following as covariates: sex and age at the time of the initial demyelinating event, serum IL-10 levels, CSF WBC count, the presence of oligoclonal bands, number of T2 lesions on the initial brain and spinal cord MRI, presence of Gd+ lesions, and EDSS during the first attacks. Dependent variables were first tested in a univariate analysis and included in a multivariate model at a threshold of 0.2  when found to be significant. The time from the initial demyelinating event to relapse was estimated by Kaplan–Meier survival analyses. Log-rank tests were used to compare survival data between low and high serum IL-10 levels. A two-tailed Spearman's rank correlation coefficient was used to ascertain the associations. The significance level was established at n = 46, 66.7%). Of all 69 patients, the affected regions were as follows: optic nerve, 16 (23.2%) patients; brainstem, 21 (30.4%) patients; spinal cord, 43 (62.3%) patients; and cerebrum, 20 (29.0%) patients. No patients were positive for AQP4-Ab in serum when the first attacks occurred. All patients received corticosteroids during acute relapse after serum samples were collected. The mean follow-up time of patients with CIS was 23.3 ± 8.2 months. The median follow-up time was 23.1 (9.5–39.9) months. There was at least 1 year of follow-up for 91.3% (n = 63) of patients and 2 years for 46.4% (n = 32). Of 69 patients, 20 (29.0%) had second events during follow-up at a mean time of 15.3 ± 9.9 months. Characteristics of CIS patients who relapsed during follow-up are presented in p = 0.041). Of 20 CIS-R patients, seven converted to MS according to the 2010 McDonald's Diagnostic Criteria; eight converted to NMOSD according to the 2015 Revised International Criteria; three patients were diagnosed with relapsing myelitis; one with relapsing brainstem encephalitis; and one with tumefactive demyelination. Of the eight NMOSD patients, only one patient was positive for serum AQP4-Ab at relapse.A total of 69 patients were included in the study. The patient screening process is shown in p = 0.015, p < 0.001, and p = 0.022, respectively; First, we compared serum cytokines levels between the entire cohort of CIS patients and control subjects with OND. The IL-23, IL-10, and TGF-β1 levels were significantly higher in patients with CIS than in patients with OND  for serum IL-10 were significant for relapse , CSF WBC count >5.5 /ul , and Gd+ lesions presence in baseline MRI  were associated with an increased risk of relapse. In the multivariate analysis, only the serum IL-10 < 1.3 pg/ml  was predictive of relapse.p = 0.001).We next examined the time to relapse in patients with high and low serum IL-10 levels using Kaplan–Meier survival analysis. As depicted in p = 0.035, p = 0.036, and p = 0.049, respectively; Comparing the low IL-10 and high IL-10 groups, we found significantly higher counts of CSF WBC, T2 lesions, and Gd+ lesions in baseline MRI in the low IL-10 group than those of the high IL-10 group  using the Spearman rank correlation test. However, we were unable to find any significant correlations between serum IL-10 levels and these data.p = 0.026). There were no significant differences in other baseline variables between the two groups.The baseline characteristics of CIS patients who converted to NMOSD (CIS-N) and MS (CIS-MS) are summarized in No significant differences were found in any cytokine levels between CIS patients who converted to NMOSD and those who converted to MS.Prospective studies demonstrate that 60–70% of CIS patients develop a second clinically evident demyelinating event within 20 years , 12 and As a key immunosuppressive cytokine, IL-10 is expressed by cells of the innate and the adaptive immune system, including dendritic cells (DCs), macrophages, mast cells, natural killer (NK) cells, eosinophils, neutrophils, CD4 and CD8 T cells, and B cells [reviewed in –15]. IL-+CD1dhi B cells as well as a better clinical outcome  is considered a severe variant of MS, and it is frequently misdiagnosed as such. The discovery of AQP4-Ab in 2004 greatly contributed to differentiating CIS from MS, revolutionizing diagnosis , 39. In As CIS can convert to different IIDDs, future research is necessary for investigating factors affecting the conversion of these disorders and their predictive biomarkers. In this study, we found no significant differences in any cytokine levels between CIS patients who converted to NMOSD compared with those who converted to MS. However, in previous researches, the serum cytokine profiles of NMOSD patients were distinct from those with MS , 45–47. None of the CIS patients were positive for AQP4-Ab in serum at the first attack. This may be because AQP4-Ab-positive patients can be diagnosed with NMOSD as only one core clinical characteristic is needed, so few patients diagnosed with CIS remain. AQP4-Ab is known to be pathogenic and can be detected as early as a decade prior to the onset of the disease –50. SeveSome limitations in our study need to be addressed. Because our patient cohort was small and derived from a single university hospital, and the follow-up duration was short, the results should be verified in larger-scale, multicenter studies with long-term follow-up. We could not adequately analyze the serial change in serum cytokine levels after first onset, and whether this change would affect the risk of recurrence and conversion could not be determined. Also the anti-MOG antibodies were not measured in our study. Further studies are expected to address these issues. In addition, it is necessary to investigate the role of the IL-10 pathway in CIS pathogenesis and therapeutic strategies.The aggregate results from this study confirm that serum IL-10 serves as a prognostic biomarker associated with the risk of relapse and the time to second events in CIS patients. We propose that serum IL-10 levels should be measured in CIS patients to identify those individuals with a high risk of relapse. This result suggests that reinforcement of the IL-10 pathway may be a promising therapeutic strategy for patients at high risk of relapse.The study was approved by the Ethics Committee of Beijing Tiantan Hospital Affiliated to Capital Medical University, Beijing, People's Republic of China (No. KY2015-031-02), and written informed consent was obtained from all participants.YW, XZ, and LY conceived and designed the study. HC performed the MILLIPLEX® map human High Sensitivity Cytokine Panels tests and ELISA tests. YW, HF, and XL participated in evaluating the EDSS scores of patients and collecting blood samples. YW performed the statistical analysis. XZ and LY revised the manuscript. All authors reviewed the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of this study was to evaluate the efficacy of denosumab or zoledronic acid (ZA) using symptomatic skeletal events (SSEs) as the primary endpoint in Asian postmenopausal women with oestrogen-receptor-positive advanced breast cancer.Asian postmenopausal women with oestrogen-receptor-positive advanced breast cancer receiving subcutaneous denosumab 120 mg Q4W, or intravenous ZA 4 mg Q4W until the primary analysis cut-off date were retrospectively analysed in the Hong Kong Practice-Based Cancer Research Center(HKCRC) from March 2011 to March 2013. The time to first on-study SSE that was assessed either clinically or through routine radiographic scans was the primary endpoint.n = 120, mean age of 64.9 years (SD 3.01) and n = 122, 65.4 years (3.44), respectively). The median times to first on-study SSE were 14.7 months (12.9–45.6) and 11.7 months (9.9–45.6) for denosumab and ZA, respectively . Compared with the ZA group, denosumab-treated patients had a significantly delayed time to first SSE . An increased incidence of SSE was found in the 16-month follow-up with rates of 2.1 and 10.7% for denosumab and ZA, respectively (P = 0.033). The difference persisted with time with rates of 8.3 and 17.2% at the final follow-up, respectively (P < 0.05).242 patients received denosumab or ZA treatment (In postmenopausal women aged ≥60 years with oestrogen-receptor-positive advanced breast cancer, denosumab significantly reduced the risk of developing SSEs compared with ZA. The findings of this pilot trial justify a larger study to determine whether the result is more generally applicable to a broader population. Oestrogen-receptor-positive advanced breast cancer, one of the most common malignancies reported worldwide, has a protracted risk of symptomatic skeletal events(SSEs), which are defined as radiation to the bone, symptomatic pathologic fracture, surgery to the bone, or symptomatic spinal cord compression –4. SSEs Denosumab, a fully human monoclonal antibody, neutralizes the Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL), which is essential for the formation, function, and survival of the osteoclasts; also, it blocks osteoclast-mediated bone resorption , 9. GnanFor Asian postmenopausal women with oestrogen-receptor-positive advanced breast cancer, to the best of our knowledge, a direct comparison between denosumab and ZA has rarely been reported in the published literature. Denosumab and ZA are effective interventions to prevent SSEs, and they provide a new bone protection option for postmenopausal women with oestrogen-receptor-positive advanced breast cancer. Nevertheless, several unanswered questions remain . The purSubjects included in this analysis were collected from the Hong Kong Practice-Based Cancer Research Center (HKCRC). Eligible patients were Asian postmenopausal women aged ≥60 years with histologically or cytologically confirmed oestrogen-receptor-positive advanced breast cancer diagnosed from March 2011 to March 2013. All patients had completed treatment with surgery and/or radiation and chemotherapy ≥4 weeks before study enrolment and received adjuvant denosumab or ZA therapy. The main exclusion criteria were the following: previous denosumab or ZA exposure or any aromatase inhibitor use during the 3 months; non-healed or planned surgery; chemotherapy or endocrine therapy for other diseases; active metabolic bone disease; undergoing castration; recurrence and metastasis before SSEs; use of medication that affects bone metabolism; renal deterioration during treatment; other primary tumours or advanced cancer; discontinuation or interruption of denosumab or ZA treatment; unable to complete surveys; life expectancy < 1 year; a BMD T score ≤ − 2.5 SD at the lumbar spine or femoral neck; modification of therapy during the follow-up period; severe infectious diseases; an American Society of Anesthesiologists (ASA) score of IV or V; and co-occurring mental illness.The patients’ body mass index (BMI) was calculated during the clinical examination. The BMD T-score was measured at the lumbar spine or femoral neck. Radiographic skeletal surveys of SSEs confirmed radiologically by a radiologist were performed within 4 weeks, every 3 months thereafter, and at the end-of-study visit. SSEs were defined as radiation therapy to the bone (including radioisotopes), pathologic fracture (excluding trauma), surgery to the bone, or spinal cord compression.This was a retrospective study in which postmenopausal women with oestrogen-receptor-positive breast cancer underwent either subcutaneous denosumab 120 mg Q4W or intravenous ZA 4 mg Q4W until the primary analysis cut-off date. The denosumab and ZA doses were not adjusted or suspended. All patients were instructed to take calcium (1 g/day) and vitamin D (400 IU/day). The same antineoplastic therapy was routinely used and was not adjusted in the two groups.p-value < 0.05 was used as a threshold for significance. All analyses were conducted using SPSS software .The descriptive and outcome variables were assessed by calculating the frequencies, means, and standard deviations (SDs). Comparison between groups was conducted using Pearson’s Chi-square test or the Mann–Whitney U test for continuous variables. The time to first SSE was assessed using a Kaplan-Meyer analysis and Log-rank test. The risk and factors of SSEs were analysed by the Cox regression model. The analyses were based on the data up to the primary analysis cut-off. A n = 120, mean age 64.9 years (SD 3.01) and ZA group n = 122, 65.4 years (SD 3.44), respectively) met the inclusion criteria. Details are shown in Fig. p = 0.0002). Compared with the ZA group, denosumab-treated patients had a significantly delayed time to first SSE . An increased incidence of SSE was found at the 16-month follow-up with rates of 2.1 and 10.7% for denosumab and ZA, respectively (P = 0.033). The difference persisted with time with rates of 8.3 and 17.2% at the final follow-up (P < 0.05).A total of 966 patients were assessed for study eligibility. Of these, 242 patients  (Table P = 0.04). The incidence of first on-study SSEs was 5.8% (7/120) after 2 years, which was also significantly lower than that in the ZA group (P = 0.04), but there was no significant difference between groups in the first on-study SSE within the 2-year period .In the ZA group, the first on-study SSE incidence was 17.2% (21 /122), in which the first on-study SSE incidence during 2-year drug use was 3.3% (4/122). After 2-year treatment, 13.9% (17/122) of cases developed a first on-study SSE. In contrast, in the denosumab group, the incidence of first on-study SSEs was only 8.3% (10/120), which was lower than in the ZA group (P = 0.002). The median time to the first on-study SSE was not reached in the denosumab group, while it was 25.5 months in the ZA group. As previously reported, denosumab reduced the risk of first on-study SSEs by 18% compared to ZA. The risk of a first on-study SSE in the ZA group was 10 times higher than that in the denosumab group during the 2-year drug use . After 2-year treatment, the ZA group had an increased risk of a first on-study SSE compared with the denosumab group .Compared with ZA, denosumab reduced the risk of first on-study SSEs by 20%  , increased the trend in the first on-study SSE risk, but no significant differences were observed (P > 0.05) . The risk of a first on-study SSE with a family history of SSEs was approximately 2 times higher than for someone with no family history of SSEs . The risk of a first on-study SSE in a denosumab-treated patient aged > 60 years was approximately 1-fold higher than in a ZA-treated patient . The clinical risk of other factors that influence the occurrence of SSEs, including menopausal patients aged ≤50 years and low BMI  and the time to the first and subsequent on-study SSEs . Martin et al. [Few previous reports have focused on denosumab versus ZA for preventing SSEs in Asian postmenopausal women with oestrogen-receptor-positive breast cancer in the Asian population –26. Howen et al.  evaluaten et al. , 22, andn et al. , 22.Throughout the follow-up period, the risk of SSEs in the denosumab group was nearly 7 times lower than that in the ZA group. However, there was no significant difference in the risk of SSEs within the 2-year period. Some studies claimed that ZA-treated patients had an increased trend in the risk of SSEs compared with denosumab-treated patients , 30, whiThe clinical risk factors  in the two groups were analysed in the current study. We found that patients with a history of fracture or a family history of fractures had a higher risk of SSEs. The age of menopause, BMI and chemotherapy history had no significant effect on the occurrence of SSEs; the specific reasons for this finding are unclear and may be related to bone.The limitations of the study include the short follow-up period, small sample size, and lack of follow up evaluation of the changes in the blood calcium and BMD. Nevertheless, the patient population is a strength of the study and suggests that the current findings are applicable to a wide range of postmenopausal women with oestrogen-receptor-positive breast cancer. Additionally, the precision for estimating the percent of SSEs is low, resulting in large confidence intervals. Although the study is powered to detect SSE differences, the analyses in this study failed to exclude subtle fractures that occurred before the endpoint measurement.At the time of the cut-off date for this analysis, we conclude, as expected, that denosumab significantly reduced the risk of SSEs compared with ZA in Asian postmenopausal women who had oestrogen-receptor-positive breast cancer regardless of whether they were followed for 2 years or less, which provided further evidence for the superior ability of denosumab to prevent SSEs versus ZA. Further investigation of the long-term utility of denosumab and ZA is necessary.